Glucocorticoid-resistant variants of mouse thymic lymphosarcoma have been selected in vivo and in culture and have been compared with respect to glucocorticoid-binding and hormone-response properties. Variants selected in vivo are similar to wild type with respect to total glucocorticoid binding and extent of nuclear translocation of the hormone-receptor complex. Cell culture lines established from such resistant populations exhibit slightly reduced (about 30%) numbers of dexamethasone binding sites. All variants selected in vivo are completely sensitive to the antiproliferative effects of 10(-7)M dexamethasone in culture. We propose that different mechanisms give rise to glucocorticoid resistance in vivo and in culture.